Inhibits Heat Shock Research Articles

Computational and experimental characterization of isomers of escin-induced renal cytotoxicity by inhibiting heat shock proteins

Escin is a natural mixture of triterpene saponins, exhibits anti-oedematous properties and promotes venous drainage by oral administration or injection. Upon clinical application of escin, adverse kidney reactions have been reported and the nephrotoxic mechanism responsible for this reaction remains elusive. In the present study, four isomeric escins (β-form: escin Ia and escin Ib; α-form: isoescin Ia and isoescin Ib) were found severely decreasing the cell viability of human kidney (HK-2) cells. A decline in HK-2 cell viability caused by sodium aescinate (a mixture of four isomers) was reduced after β-glucuronidase hydrolysis. In addition, sodium aescinate concentration-dependently inhibited the expression level of heat shock proteins (HSPs) in the Madin-Darby Canine Kidney (MDCK) cells. Moreover, with molecular docking and molecular dynamics simulation, these four isomeric escins could directly bind to the ATP-binding domain of HSP70 and HSP90, thus competitively inhibiting the function of HSPs. Escin Ia is bound to HSPs with the lowest binding free energy, which is consistent with the observation that escin Ia most severely decreases HK-2 cell viability. Thus, we demonstrate a heretofore unknown molecular mechanism of escin-induced renal cytotoxicity as well as identify HSPs as potential targets for the renal cytotoxic effect of escin.

PI3Kδ/AKT Inhibition Augments 2-Phenylethynesulfonamide Induced Apoptosis Via the Unfolded Protein Response in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is largely comprised of two major subsets, IGHV unmutated (U-CLL) and mutated (M-CLL) CLL, associated with progressive and indolent disease respectively. Approximately 15% patients harbour a 17p deletion and/or a TP53 mutation which is largely associated with the worst clinical outcome. The development of BCR signalosome inhibitors such as ibrutinib and idelalisib have been instrumental in the treatment of CLL, however these drugs are not curative, only supress the disease and resistance is already developing particularly in patients with a complex karyotype associated with 17p del/ TP53 mutation. Consequently continued development of novel therapies targeting pathways which enhance selective killing of tumor cells independently of p53 are urgently required. We previously demonstrated that 2-phenylacetylenesulfonamide (PAS), induced apoptosis of CLL cells in a p53-independent manner. PAS is suggested to inhibit heat shock protein (HSP) 70, a key protein involved in protein folding. Therefore in CLL cells, PAS would be predicted to cause cellular stress due to the accumulation of misfolded proteins resulting in the activation of the unfolded protein response (UPR). The UPR serves to protect cells from the consequences of protein misfolding and can function in part by activating pro-survival signaling, including the PI3K/AKT pathway. Under normal conditions the ER chaperone BiP inhibits activation of the UPR by binding the three ER transmembrane receptors; inositol-requiring enzyme 1 (IRE-1α), pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Activation of the UPR leads to the upregulation of HSP family members including HSP70 in the cytosol and BiP/GRP78 in the endoplasmic reticulum (ER). These HSPs aid in refolding misfolded proteins or facilitate their degradation via the proteasome. If the stress is resolved, the UPR signalling pathway is switched off; however, if stress persists the UPR remains active and switches from an anti-apoptotic to a pro-apoptotic signal via p38MAPK.Aim: To determine the mechanism of PAS induced apoptosis in CLL cells.Methods: Fifty two CLL samples were treated with PAS (5-20μM) and cellular responses assessed using immunoblotting, flow cytometry and MTT assay.Results: In this study we demonstrated that the UPR is induced in CLL cells following the induction of reactive oxygen species by PAS treatment. This consequently led to the activation of both pro-apoptotic (Noxa, p<0.0001) and anti-apoptotic (AKT, p<0.05) signalling pathways. PAS treatment further increased expression of key stress proteins BiP (p=0.0005) and HSP70 (p=0.001). PAS treatment also resulted in activation of the PERK and IRE-1α arms of the UPR (cleaved XBP-1, phosphorylated eiF2α, phosphorylated p38MAPK) within 2h, which persisted up to 24h. This pathway was pivotal for CLL cell death since small molecule inhibition of either p38MAPK or PERK signalling significantly reduced PAS mediated apoptosis by approximately 50%. However PAS induced apoptosis was simultaneously antagonised by activation of the pro-survival AKT pathway. Consequently we hypothesised that PAS treatment in combination with a PI3Kδ (idelalisib) or AKT (AKTi) inhibitor may promote greater CLL cell apoptosis. Indeed our combination indices (CI) studies demonstrated that simultaneous treatment with an AKTi or the clinically approved PI3Kδ inhibitor idelalisib and PAS synergistically and significantly enhanced apoptosis of CLL cells compared with either agent alone (p<0.05).Conclusion: This data suggests that inhibiting the pro-survival arm of the UPR (PI3K/AKT), whilst simultaneously activating the pro-death arm (HSP70/p38MAPK), may be a useful strategy for the treatment of CLL and particularly samples with a 17p del or TP53 mutation and warrants further clinical investigation particularly in CLL cases that are resistant to BCR kinase or Bcl-2 family inhibitors. DisclosuresStrefford:Roche: Research Funding. Johnson:Novartis: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Carrick: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Karus: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Zenyaku Kogyo: Consultancy, Honoraria; Epizyme: Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele:Gilead: Consultancy, Honoraria; Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress.

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

Discovery of an orally active antitumor agent that induces apoptosis and suppresses EMT through heat shock protein 90inhibition.

Background Nowadays, lung cancer seriously affects human health in the world. Therefore, it is of great significance to develop effective anti-lung cancer drugs. Methods In this work, chalcone derivative HYQ97 was designed via a molecular hybridization strategy. It was synthesized by the cycloaddition in the presence of sodium ascorbate under mild conditions. Lung cancer cell lines were cultured to investigate its antitumor effects in vitro and in vivo. Results HYQ97 inhibited the proliferation of lung cancer cell lines. Specifically, its IC50 value against lung cancer A549 cells was 74.26 nM. It could inhibit heat shock protein 90 (Hsp90) and degrade its client proteins in a dose-dependent manner. Furthermore, HYQ97 suppressed the epithelial mesenchymal transition process and induced apoptosis of A549 cells. Importantly, HYQ97 also had significant inhibitory effects on tumor growth in vivo. Conclusions Chalcone derivative HYQ97 is a promising candidate for lung cancer treatment.

Paclitaxel increases sensitivity of SKOV3 cells to hyperthermia by inhibiting heat shock protein 27

Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising treatment strategy for patients with peritoneal metastasis of ovarian cancer. Heat shock proteins (HSPs) play an important role in cellular stress during HIPEC treatment. The aim of the present study was to investigate whether paclitaxel can exert antitumor effects by inhibiting heat shock protein 27 (HSP27) expression during HIPEC treatment. Cell viability was detected by CCK8 assay. We used Western blot analysis to detect HSP27 expression under hyperthermia conditions with or without paclitaxel in SKOV3 cells. To further examine the role of HSP27 in the apoptosis, Bcl-2, Bax and Caspase-3 protein expression were additionally determined after reducing HSP27 levels using an siRNA strategy, and apoptosis was detected using the Annexin V/PI assay. The upregulation of HSP27 expression was accompanied with a rise in temperature. In addition, HSP27 could promote Bcl-2 expression, inhibit Bax and Caspase-3 expression, reduce the Bax / Bcl-2 ratio markedly in SKOV3 cells. Furthermore, paclitaxel could upregulate the Bax / Bcl-2 ratio by inhibiting HSP27 expression, and in turn, promoting apoptosis due to hyperthermia. Paclitaxel could also promote apoptosis by inhibiting HSP27 in SKOV3 cells. Our results demonstrate a synergistic effect between paclitaxel and hyperthermia at the cellular level.

Pathological Mechanism of Photodynamic Therapy and Photothermal Therapy Based on Nanoparticles.

The ultimate goal of phototherapy based on nanoparticles, such as photothermal therapy (PTT) which generates heat and photodynamic therapy (PDT) which not only generates reactive oxygen species (ROS) but also induces a variety of anti-tumor immunity, is to kill tumors. In addition, due to strong efficacy in clinical treatment with minimal invasion and negligible side effects, it has received extensive attention and research in recent years. In this paper, the generations of nanomaterials in PTT and PDT are described separately. In clinical application, according to the different combination pathway of nanoparticles, it can be used to treat different diseases such as tumors, melanoma, rheumatoid and so on. In this paper, the mechanism of pathological treatment is described in detail in terms of inducing apoptosis of cancer cells by ROS produced by PDT, immunogenic cell death to provoke the maturation of dendritic cells, which in turn activate production of CD4+ T cells, CD8+T cells and memory T cells, as well as inhibiting heat shock protein (HSPs), STAT3 signal pathway and so on.

Oxymatrine ameliorates imiquimod-induced psoriasis pruritus and inflammation through inhibiting heat shock protein 90 and heat shock protein 60 expression in keratinocytes

In this work, we aimed to investigate whether oxymatrine exerts its anti-pruritic and anti-inflammatory efficacy in the imiquimod-induced psoriasis mice and the related mechanism. We established the psoriasis model by applying the imiquimod ointment topically and oxymatrine was injected intraperitoneally as the treatment. The behavior and skin morphology results indicated that oxymatrine inhibits imiquimod-induced pruritus alleviating keratinization of skin and inflammatory infiltration. Moreover, we examined the expression of various indicators and found heat shock protein (HSP) 90 and 60 upregulated in model group, which were reversed in oxymatrine treated groups. Molecular docking and the studies in vivo confirmed that HSP90 and HSP60 participate in the inhibitory effect of oxymatrine on the phenotypes of psoriasis mice. Mechanically, immunofluorescence staining demonstrated that oxymatrine-induced downregulation of HSP90 and HSP60 was mainly in keratinocytes. In vitro results showed that oxymatrine decreases the expression of HSP90 and HSP60 upregulated by TNF-α and IFN-γ in HaCaTs cells and the siRNA mediated HSP90 and HSP60 silencing reverses inflammation inhibited by oxymatrine. Taken together, these results indicate that oxymatrine relieves psoriasis pruritic and inflammation by inhibiting the expression of HSP90 and HSP60 in keratinocytes through MAPK signaling pathway

Inhibiting Heat Shock Protein 90 Protects Nucleus Pulposus-Derived Stem/Progenitor Cells From Compression-Induced Necroptosis and Apoptosis.

Nucleus pulposus-derived stem/progenitor cells (NPSCs) provide novel prospects for the regeneration of degenerated intervertebral disc (IVD). Nevertheless, with aging and degeneration of IVD, the frequency of NPSCs markedly decreases. Excessive cell death could be the main reason for declined frequency of NPSCs, however, the exact mechanisms remain elusive. Thus, the present study was undertaken to explore the mechanisms of compression-induced NPSCs death, and the effects of heat shock protein 90 (HSP90) on NPSCs survival. Here, we found that compression could trigger receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis of NPSCs. Furthermore, we found that elevated expression of HSP90 was involved in compression-induced NPSCs death, and inhibiting HSP90 could dramatically attenuate compression-induced necroptosis of NPSCs via regulating the expression and activity of RIPK1/RIPK3/MLKL, and alleviating the mitochondrial dysfunction (mitochondrial membrane potential loss and ATP depletion) and oxidative stress [production of mitochondrial reactive oxygen species (ROS), cellular total ROS and malondialdehyde, and downregulation of superoxide dismutase 2]. Besides necroptosis, compression-induced apoptosis of NPSCs was also attenuated by HSP90 inhibition. In addition, we found that enhanced expression of HSP70 contributed to the cytoprotective effects of inhibiting HSP90. More encouragingly, our results demonstrated that inhibiting HSP90 could also mitigate the exhaustion of NPSCs in vivo. In conclusion, RIPK1/RIPK3/MLKL-mediated necroptosis participates in compression-induced NPSCs death. Furthermore, targeting HSP90 to simultaneously inhibit necroptosis and apoptosis of NPSCs might be an efficient strategy for preventing the death of NPSCs, thus rescuing the endogenous repair capacity of NP tissue.

FAM83G Is a Novel Inducer of Apoptosis.

The family with sequence similarity 83 (FAM83) protein family G (FAM83G) possesses a predicted consensus phosphorylation motif for serine/threonine-protein kinase D1/protein kinase C mu (PKD1/PKCμ) at serine residue 356 (S356). In this study, overexpressed wild-type FAM83G coimmunoprecipitated with PKD1/PKCμ in Chinese hamster ovary (CHO) cells inhibited heat shock protein 27 (HSP27) phosphorylation at S82 and reduced the living cell number. The expression of a FAM83G phosphorylation-resistant mutant (S356A-FAM83G) had no effect on the living cell number or the induction of spontaneous apoptosis. By contrast, the introduction of a synthetic peptide encompassing FAM83G S356 into HCT116 and HepG2 cells decreased HSP27 S15 and S82 phosphorylation and induced spontaneous apoptosis. On the other hand, the introduction of FAM83G phosphorylation-resistant mutant synthesized peptides (S356A-AF-956 and S356A-AG-066) did not reduce the living cell number or induce spontaneous apoptosis. The endogenous expression of HSP27 and FAM83G was apparently greater in HCT116 and HepG2 cells compared with in CHO cells. In various types of lung cancer cell lines, the FAM83G messenger RNA (mRNA) level in non-small lung cancer cells was at a similar level to that in non-cancerous cells. However, the FAM83G mRNA level in the small cell lung cancer cell lines was variable, and the HSP27 mRNA level in FAM83G mRNA-rich types was greater than that in FAM83G mRNA-normal range types. Taken together, these data demonstrate that FAM83G S356 phosphorylation modulates HSP27 phosphorylation and apoptosis regulation and that HSP27 is a counterpart of FAM83G.

Inhibitory Effect of Manassantin B Isolated from Saururus chinensis on Skin Heat Aging

Heat shock treatment-induced skin aging causes a thickened epidermis, increased matrix metalloproteinase (MMP)-1 expression, collagen degradation, and deep wrinkles. In this study, we investigated the effect of manassantin B in preventing heat shock treatment-induced aging. We first separated manassantin B (MB) from the roots of Saururus chinensis, and the structure was identified using 1H- and 13C-NMR spectroscopy. RT-PCR and western blotting were applied to investigate the anti-aging effect of manassantin B. Manassantin B decreased MMP-1 expression through transient receptor potential vanilloid (TRPV) 1 channel inhibition and significantly increased procollagen expression. In addition, manassantin B suppressed MAPK phosphorylation in a dose-dependent manner. Our results suggest that manassantin B, the active ingredient in S. chinensis, can be effectively used to inhibit heat shock treatment-induced skin aging.

Schizandrin A exhibits potent anticancer activity in colorectal cancer cells by inhibiting heat shock factor 1.

Heat shock factor 1 (HSF1) is a powerful multifaceted oncogenic modifier that plays a role in maintaining the protein balance of cancer cells under various stresses. In recent studies, there have been reports of increased expression of HSF1 in colorectal cancer (CRC) cells, and the depletion of the HSF1 gene knockdown has inhibited colon cancer growth both in vivo and in vitro. Therefore, HSF1 is a promising target for colon cancer treatment and chemoprevention. In the present study, we found that Schizandrin A (Sch A) significantly inhibited the growth of CRC cell lines by inducing cell cycle arrest, apoptosis and death. Through HSE luciferase reporter assay and quantitative PCR (qPCR), we identified Sch A as a novel HSF1 inhibitor. In addition, Sch A could effectively inhibit the induction of HSF1 target proteins such as heat-shock protein (HSP) 70 (HSP70) and HSP27, whether in heat shock or normal temperature culture. In the Surface Plasmon Resonance (SPR) experiment, Sch A showed moderate affinity with HSF1, further confirming that Sch A might be a direct HSF1 inhibitor. The molecular docking and molecular dynamic simulation results of HSF1/Sch A suggested that Sch A formed key hydrogen bond and hydrophobic interactions with HSF1, which may contribute to its potent HSF1 inhibition. These findings provide clues for the design of novel HSF1 inhibitors and drug candidates for colon cancer treatment.

Conserved microRNA mediates heating tolerance in germ cells versus surrounding somatic cells

ABSTRACT Mammalian fertility is reduced during heat exposure in the summer, but is regained as temperatures decrease in the autumn again. However, the mechanism underlying the phenomenon remains unknown. We investigated heat stress tolerance of germ cells and their surrounding somatic cells, and discovered that microRNA ssc-ca-1 was upregulated after heat stress in cultured porcine granulosa cells (GCs), but not in serum-starved GCs. Ssc-ca-1 inhibited heat shock protein 70 (Hsp70) expression through its 3′- and 5′-UTRs. Although Hsp70 mRNA transcription was induced in GCs by in vivo exposure to heat in the summer, ssc-ca-1 inhibited Hsp70 expression. In ovarian cultures, heat stress-induced Hsp70 expression in primordial but not in growing follicles; ssc-ca-1 expression did not change in primordial follicles, but increased in growing follicles. Consistently, ssc-ca-1 was present in testicular cells and exhibited the same function as in ovarian cells. It modulated the different Hsp70 expression between spermatogonial stem cells and Sertoli cells after scrotal heat stress. This mechanism is of relevance to mammalian fertility in parts of the world dominated by heat stress associated with global climate change.

The succinct synthesis of AT13387, a clinically relevant Hsp90 inhibitor

AT13387 is an orally bioavailable clinical candidate developed to inhibit heat shock protein 90 (Hsp90). This article describes a modified synthetic route for the multi-gram production of AT13387 in 46% overall yield. The modified synthetic route is short, avoids stringent reaction conditions and difficult purifications, which led to an increase in overall yield.

Gambogic acid attenuates liver fibrosis by inhibiting the PI3K/AKT and MAPK signaling pathways via inhibiting HSP90

Gambogic acid (GA), a major ingredient of Garcinia hanburryi, is known to have diverse biological effects. The present study was designed to evaluate the anti-fibrotic effects of GA on hepatic fibrosis and reveal its underlying mechanism. We investigated the anti-fibrotic effect of GA on dimethylnitrosamine and bile duct ligation induced liver fibrosis in rats in vivo. The rat and human hepatic stellate cell lines (HSCs) lines were chose to evaluate the effect of GA in vitro. Our results indicated that GA could significantly ameliorate liver fibrosis associated with improving serum markers, decrease in extracellular matrix accumulation and HSCs activation in vivo. GA significantly inhibited the proliferation of HSC cells and induced the cell cycle arrest at the G1 phase. Moreover, GA triggered autophagy at early time point and subsequent initiates mitochondrial mediated apoptotic pathway resulting in HSC cell death. The mechanism of GA was related to inhibit heat shock protein 90 (HSP90) and degradation of the client proteins inducing PI3K/AKT and MAPK signaling pathways inhibition. This study demonstrated that GA effectively ameliorated liver fibrosis in vitro and in vivo, which provided new insights into the application of GA for liver fibrosis.

Accelerated evolution of lager yeast strains for improved flavour profiles

Fermented food and beverages have accompanied humans throughout history. Several species of microorganisms can transform raw materials into products with different and improved characteristics, for example alcoholic beverages (wine and beer) or dairy products, such as yogurt. In a world of constant change and competitiveness, brewers have to modulate and create new products to satisfy consumers. Therefore, the aim of my project is to generate lager yeast capable of producing molecules that improve the organoleptic profile of alcoholic beverages and understand the genetic and biochemical changes that increases the production of these aromatic molecules. The initial work has focused on the Ehrlich Pathway and the ester production. To produce changes in the genome, two approaches have been followed: one chemical way, using Radicicol, and one physical way, Heat Shock Thermal Stress (HSTS). Both ways inhibit Heat Shock proteins. Furthermore, this inhibition produces DNA damage, introducing Single sStrand Breaks (SSBs) and Double Strand Breaks (DSBs). The result of this inhbition is damage in the DNA and, furthermore, mutations in the DNA (aneuplidies and INDELS). After the Accelerated evolution, the evolved strains were plated on media containing amino acid analogues. These amino acid analogues select for cells with decreased feed-back inhibition of amino acid biosynthesis. Different mutant strains were generated with these two ways. These strains will be tested by different approaches, as growth curves, genome ploidy using cCGH and CHEF gel and qPCR analysis to measure the flux throughout the fermentation.

Correction: Flower-like gold nanoparticles for enhanced photothermal anticancer therapy by the delivery of pooled siRNA to inhibit heat shock stress response.

Correction for 'Flower-like gold nanoparticles for enhanced photothermal anticancer therapy by the delivery of pooled siRNA to inhibit heat shock stress response' by Yanan Liu et al., J. Mater. Chem. B, 2019, 7, 586-597.

Flower-like gold nanoparticles for enhanced photothermal anticancer therapy by the delivery of pooled siRNA to inhibit heat shock stress response.

Due to the anti-apoptotic effect employed by cells to protect themselves, recent research shows that photothermal therapy (PTT) can lead to heat shock response, thus reducing the effect of treatment on cancer cells. Small interfering RNA (siRNA), as an effective carrier of RNA interference, can silence the expression of heat shock protein, HSPs or BAG3 genes by inhibiting the expression of specific genes, and thereby inhibiting heat shock response and making cancer cells more sensitive to PTT. In this study, flower-like gold nanoparticles were used as a core for a layer-by-layer strategy to produce a safe and biodegradable nanoparticle platform for gene silencing and photothermal therapy. The results showed that when the mass ratio of the GNFs and siRNA was 20 : 1, the loading efficiency was above 90%, which can effectively silence the expression of BAG3 siRNA. We demonstrated that the GNFs-siRNA still had a good photothermal effect after siRNA modification. In vitro, the GNFs-siRNA showed good biocompatibility and effectively tumor killing properties after laser irradiation. Furthermore, the GNFs-siRNA with laser treatment significantly decreased the expression of BAG3 and remarkably inhibited tumor growth in vivo. This nanosystem establishes an optimized platform for future gene delivery and photothermal therapy.

Triptolide, a HSP90 middle domain inhibitor, induces apoptosis in triple manner.

Triptolide (TL) is a potent anti-tumor, anti-inflammatory and immunosuppressive natural compound. Mechanistic studies revealed that TL inhibits tumor growth and triggers programmed cell death. Studies further suggested that TL inhibits heat shock response in cancer cells to induce apoptosis. HSP90β is the major component of heat shock response and is overexpressed in different types of cancers. Given almost all identified HSP90β inhibitors are either N or C-terminal inhibitors, small molecules attacking cysteine(s) in the middle domain might represent a new class of inhibitors. In the current study, we showed that TL inhibits HSP90β in triple manner. Characterization suggests that TL inhibits ATPase activity by preventing ATP binding thus blunts the chaperone activity. TL disrupts HSP90β-CDC37 (co-chaperone) complex through middle domain Cys366 of HSP90β and causes kinase client protein degradation. At the cellular level, the TL-mediated decrease in CDK4 protein levels in HeLa cells causes reduced phosphorylation of Rb resulting in cell cycle arrest at the G1 phase. Furthermore, our results demonstrated that TL triggers programmed cell death in an HSP90β-dependent manner as knockdown of HSP90β further sensitized TL-mediated cell cycle arrest and apoptotic effect. Surprisingly, our data showed that TL is the first drug to be reported to induce site-specific phosphorylation of HSP90β to drive apoptosome formation in the early phase of the treatment.In summary, our study established that TL is a novel middle domain HSP90β inhibitor with bi-phasic multi-mechanistic inhibition. The unique regulatory mechanism of TL on HSP90β makes it an effective inhibitor.

Converting polar cyclic peptides into membrane permeable molecules using N‐methylation

AbstractDescribed are the design, synthesis, and biological evaluation of 5 N‐methylated analogs that are based on a lead drug structure LB51. LB51 is a cyclic pentapeptide that inhibits heat shock protein 90 and although a potent inhibitor of the protein function, it has poor cell permeability. Introduction of an N‐methyl moiety at each amino acid produces 5 analogs of LB51, where all 5 show significantly improved membrane permeability over the lead molecule despite the presence of 4 highly polar side chains.

Triptolide reverses apatinib resistance in gastric cancer cell line MKN45 via inhibition of heat shock protein 70

Objective: To investigate the effect of triptolide, a specific inhibitor of heat shock protein 70 (HSP70), on apatinib resistance in gastric cancer cells line MKN45. Methods: The apatinib-resistant cells (MKN45/AR) and MKN45 parental cells were treated with apatinib, triptolide and apatinib combined with triptolide, respectively. CCK-8 assay was performed to determine the half maximal inhibitory concentration (IC(50)) of MKN45/AR and MKN45 cells in the presence of different treatment. The mRNA expression of heat shock protein gene (HSPA1A and HSPA1B) was detected by RT-PCR, while the protein expression of heat shock protein 70 was analyzed using Western blot in MKN45/AR and MKN45 cells. Results: The IC(50) values of apatinib-sensitive and apatinib-resistant MKN45 cells were 10.411 μmol/L and 70.527 μmol/L, respectively, showing a significant difference (P<0.05). The mRNA expression of HSPA1A and HSPA1B in MKN45/AR cells was significantly higher than that in MKN45 cells (P<0.001). The protein expression of heat shock protein 70 was significantly decreased after 0.25 μmol/L triptolide treatment in MKN45/AR cells (P<0.01). When heat shock protein 70 was inhibited by triptolide, the IC(50) value of apatinib in MKN45/AR cells was reduced to 11.679 μmol/L, which was significantly lower than cells treated with apatinib alone (P<0.05). Conclusions: The apatinib-resistant MKN45 cells have high levels of heat shock protein 70. Low doses of triptolide can significantly inhibit heat shock protein 70, leading to reverse the resistance phenotype of MKN45/AR cells. Therefore, inhibition of heat shock protein 70 provides a new therapy strategy for patients with apatinib resistance.